Disruption of fibronectin matrix affects type IV collagen, fibrillin and laminin deposition into extracellular matrix of human trabecular meshwork (HTM) cells

Abstract

Fibronectin fibrils are a major component of the extracellular matrix (ECM) of the trabecular meshwork (TM). They are a key mediator of the formation of the ECM which controls aqueous humor outflow and contributes to the pathogenesis of glaucoma. The purpose of this work was to determine if a fibronectin-binding peptide called FUD, derived from the Streptococcus pyogenes Functional Upstream Domain of the F1 adhesin protein, could be used to control fibronectin fibrillogenesis and hence ECM formation under conditions where its expression was induced by treatment with the glucocorticoid dexamethasone. FUD was very effective at preventing fibronectin fibrillogenesis in the presence or absence of steroid treatment as well as the removal of existing fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD also disrupted the incorporation of type IV collagen, laminin and fibrillin into the ECM. The effect of FUD on these other protein matrices, however, was found to be dependent upon the maturity of the ECM when FUD was added. FUD effectively disrupted the incorporation of these other proteins into matrices when added to newly confluent cells that were forming a nascent ECM. In contrast, FUD had no effect on these other protein matrices if the cell cultures already possessed a pre-formed, mature ECM. Our studies indicate that FUD can be used to control fibronectin fibrillogenesis and that these fibrils play a role in regulating the assembly of other ECM protein into matrices involving type IV collagen, laminin, and fibrillin within the TM. This suggests that under in vivo conditions, FUD would selectively disrupt fibronectin fibrils and de novo assembly of other proteins into the ECM. Finally, our studies suggest that targeting fibronectin fibril assembly may be a viable treatment for POAG as well as other glaucomas involving excessive or abnormal matrix deposition of the ECM.

Figure 1. Proposed model for fibronectin matrix assembly

A) Fibronectin, which is secreted as a globular dimer, binds an integrin on the cell surface (step 1). Contractile forces generated by the actomyosin cytoskeleton (step 2) cause the fibronectin dimer to unfold which exposes fibronectin-fibronectin binding sites (yellow ovals) that mediate fibril formation (step 3). Eventually these fibronectin fibrils are incorporated into the extracellular matrix as a deoxycholic (DOC) acid-insoluble fibril (step 4). B) Fibronectin schematic highlighting the various domains involved in fibril formation. FUD binding to the amino terminus disrupts the intermolecular interactions involved in fibril formation. The domains labeled A, B and IIICS represent the 3 alternatively-spliced domains of fibronectin. The RGD site indicated in the 10^th type III repeat is the main integrin-binding site.

Figure 2. Recombinant FUD binds to fibronectin and disrupts fibronectin fibrillogenesis

A) Recombinant FUD (lane 2) was purified and analyzed by PAGE. Molecular weight markers are in lane 1. The recombinant FUD peptide migrated as a single band corresponding to a molecular weight of ~ 20 kDA which is larger than its theoretical size but consistent with an earlier report of FUD (Ozeri et al., 1996). B) Confluent HTM cells were left untreated (top row) or treated with 2 µM FUD for 7 days prior to fixation and double labeling with the fibronectin polyclonal antisera (FN pAb) and mAb IST-9 against EDA+ FN. C) FUD conjugated to AlexaFluor^®488 was added to confluent HTM cultures for forty-eight hours prior to fixation and labeled with either a fibronectin antisera (top row) or mAb IST-9 (bottom row). D) FUD cytotoxicity was assessed using the MTT assay in HTM cells that were plated into 96 well plates and grown to confluence. They were left either untreated (NT) or treated for 7 days with 2 µM FUD. There was no significant difference between untreated and FUD-treated cells. Scale bars = 50 µm.

Figure 3. Inhibition of fibronectin fibrillogenesis by FUD is specific

A) FUD was mutated using site-directed mutagenesis to alter key amino acids found within the C-terminal portion of the peptide in order to abrogate the activity the peptide. The bottom sequence is wild type FUD while the mutant FUD (mFUD) sequence is on top. The mutated amino acids are marked with asterisks. The underlined wild type sequences in bold are critical for FUD binding to fibronectin. The numbers 2, 3, 4, 5 & 8 indicate the fibronectin type I domains that the underlined sequences bind to (N-terminal to C-terminal) (Ma et al., 2015). B) HTM cells were plated at 25,000 cells/well into 96 wells plates and allowed to attach for 3 h. Cells were then left either untreated (NT) or treated with 0.5, 1.0, 2.0 or 4.0 µM mFUD or FUD for 24 h. Cell layers were then extracted with 1% DOC (top graph) or left un-extracted (bottom graph) prior to processing for OCW analysis. For both graphs, all FUD-treated groups are significantly less than untreated cells, p < 0.0001.

Figure 4. FUD blocks steroid-induced increases in fibronectin fibrillogenesis and matrix assembly

A) Confluent cell monolayers were treated with vehicle (EtOH) or 500 nM DEX for 12–14 days in the presence or absence of 2 µM FUD prior to fixation and double labeling with fibronectin antisera (FN pAb) and mAb IST-9 against EDA+ FN. B) Cell monolayers in 96 wells plates were treated as in panel A. We used the fibronectin polyclonal antisera in this assay rather than mAb IST-9 as the antisera would detect the multiple isoforms of fibronectin present in our HTM cultures as suggested by the immunofluorescence microscopy results described in panel A. At the end of the treatment period, cell monolayers were extracted with 1% DOC prior to processing for OCW analysis. *, significantly greater than EtOH-treated cells, p < 0.01; **, significantly less than DEX (100nM) treated cells without FUD, p < 0.05; ***, significantly less than DEX (500nM) treated cells without FUD, p < 0.01. Scale bars = 50 µm.

Figure 5. Co-localization of ECM proteins expressed by HTM cells

HTM cells that had been maintained at confluence for 7 days were double-labeled for type IV collagen and EDA+ fibronectin (top row), fibronectin and laminin (2^nd row) or type IV collagen and laminin (3^rd row). Arrows indicate areas of co-localization for the different antibody pairs. Cultures were also double-labeled with rabbit non-immune serum and a mAb against glial fibrillary acidic protein (GFAP) which served as controls for antibody specificity (4^th row). Cultures in row 1 were methanol-fixed while the other three rows show labeling in paraformaldehyde-fixed cells. Negative control labeling was also performed in methanol-fixed cells and showed similar results (not shown).

Figure 6. Relative labeling intensities of ECM proteins in newly confluent HTM cultures

HTM cells were plated into 96 wells plates and allowed to reach confluence at which point they were either fixed with 4% paraformaldehyde and processed for OCW analysis (Day 0) or maintained in culture for 7 days prior to fixation and processing (Day 7). Cell monolayers were not extracted with DOC prior to OCW analysis. A) All four proteins on the same axis. B) Blow up of the type IV collagen, laminin and fibrillin data. *, type IV collagen significantly greater at day 7 relative to day 0, p < 0.01.

Figure 7. FUD disrupts newly forming matrices of type IV collagen, laminin and fibrillin

Cells were grown to confluence at which time treatment with vehicle or 500 nM DEX in the presence of absence of 2 µM FUD was initiated. Cells were treated for 7 days prior to fixation and labeling for type IV collagen, laminin or fibrillin. Scale bars = 50 µm.

Figure 8. HTM express EDA+ and EDB+ fibronectin isoforms both of which are susceptible to disruption by FUD

Cells were grown to confluence at which time treatment with vehicle or 500 nM DEX in the presence of absence of 2 µM FUD was initiated. Cells were treated for 7 days prior to fixation and labeling with fibronectin polyclonal antisera (FN pAb), mAb IST-9 against EDA+ fibronectin or mAb BC-1 against EDB+ fibronectin. Scale bars = 50 µm.

Figure 9. Fibronectin fibrils consist of a mixture of EDB+ and EDB- fibronectin isoforms

Confluent HTM cells were fixed and labeled with fibronectin polyclonal antisera (A) and mAb BC-1 against EDB+ fibronectin (B). The merged images (C) show select regions of co-localization of the two antibodies (arrowheads). Scale bar = 50 um.

Figure 10. FUD has no effect on type IV collagen, laminin or fibrillin matrices when added to cells with a mature pre-formed ECM

Cells were grown to confluence and then allowed to form a mature ECM for 7 days prior to initiating treatment with vehicle or 500 nM DEX in the presence of absence of 2 µM FUD. Cells were treated for 12 days prior to fixation and labeling for type IV collagen, laminin or fibrillin. Scale bars = 50 µm.

Figure 11. Schematic illustrating step-wise assembly of the ECM in which fibronectin fibrils initially act as organizing centers

A) In nascent matrices, fibronectin (FN) fibrils act as a scaffold upon which other macromolecular structures are assembled. Disruption of fibronectin fibrillogenesis by FUD blocks the assembly of these other nascent protein structures. B) Addition of FUD to the mature ECM triggers the removal of existing fibronectin matrices and prevents the assembly of any new fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD under these conditions, however, does not affect laminin (LN), fibrillin (FBN) and type IV collagen (Col IV). These proteins are in macromolecular structures that now exist independently of fibronectin fibrils and are likely stabilized by their attachment to their own cell surface receptors which can include different integrins. Any newly secreted FBN, LN or Col IV (curved arrows) in these mature cultures presumably would be incorporated directly into each protein’s existing macromolecular structure.